The present invention relates to peptide nucleic acid (PNA) conjugates and a method for binding these conjugates to DNA for diagnostic and therapeutic applications. More particularly, the invention relates to PNA linked to fluorophores or peptides which hybridize to DNA and can be used to monitor the intracellular location of exogenous transfected DNA and to promote various intracellular processes.
Plasmid based, non-viral gene delivery systems have been shown to be promising for the treatment of major inherited and acquired diseases, and for the development of a new approach to vaccination (Wolff et al., Science 247:1465-1468, 1990; Ulmer et al., Science 259:1745-1749,1993; Donnelly et al., Life Sci. 60:163-172, 1997; Gao et al., Gene Ther. 2:710-722, 1995; Felgner, Ann. N.Y. Acad. Sci.772:126-139, 1995). Although the numbers of human gene therapy trials with these technologies are increasing, their efficiencies and clinical potencies are currently limited due to low levels of in vivo gene product expression (Felgner, Hum. Gene Ther.7:1791-1793, 1996). Commonly used approaches for increasing in vivo expression include improving the DNA delivery system (Gao et al., Gene Therapy 2:710-722, 1995; Felgner, supra.; Behr, Bioconj. Chem. 5:382-389, 1994) or optimizing the DNA sequence at the level of the promoter, enhancer, intron or terminator (Hartikka et al., Hum. Gene Ther. 7:1205-1217, 1996; Liang et al., Gene Therapy 3:350-356, 1996).
In conventional small molecule drug development, it is common to make systematic chemical modifications of the biologically active agent itself in order to improve its bioavailability and efficacy. This research and development activity is referred to as medicinal chemistry. The ability to carry out a medicinal chemistry approach to improve the bioavailability of DNA is presently lacking because the methods that have been employed to directly modify DNA either reduce or destroy its ability to be transcribed. In addition, the available approaches to chemically modify plasmids which involve photolysis, nick translation, or the use of chemically active nucleotide analogs, randomly attack the DNA so that the final product is chemically heterogeneous and poorly defined.
Several methodologies, including electron microscopy, fluorescence in situ hybridization (FISH), in situ polymerase chain reaction (PCR), DNA intercalating dyes and radio-, biotin-, gold-, or fluorescent-labeled DNA, have been used to follow the delivery of DNA in cells (Loyter et al., Proc. Natl. Acad. Sci. U.S.A. 79:422-426, 1982; Tsuchiya et al. J. Bacteriol. 170:547-551, 1988; Chowdhury, 1993; Zabner et al., J. Biol. Chem. 270:18997-19007, 1995; Dowty et al., Proc. Natl. Acad. Sci. U.S.A. 92:4572-4576, 1995; Bordignon et al., Science 270:470-475, 1995; Dean, Exp. Cell Res. 230:293-302, 1997). However, these methods have practical and technical limitations, which can lead to difficulties in interpretation. Electron microscopy, FISH and in situ PCR require cell to fixation, lysis, and lengthy manipulations, and these procedures have been shown to influence the detection sensitivity and pattern of DNA distribution in cells. DNA intercalating fluorescent dyes, bind weakly to plasmid and exchange with endogenous nucleic acid raising questions about the intracellular source of the fluorescent signal. Other covalent fluorescent labeling methods which utilize nick translation or photoaffinity labeling result in chemical breakdown of the starting material, and thus any observations made with these materials may not be representative of the behavior of the original intact plasmid. None of the technologies presented above allow direct detection of structurally and functionally intact plasmid in a real-time fashion in viable cells.
Peptide nucleic acids (PNA) have been developed to hybridize to single and double stranded nucleic acids. PNA are nucleic acid analogs in which the entire deoxyribose-phosphate backbone has been exchanged with a chemically completely different, but structurally homologous, polyamide (peptide) backbone containing 2-aminoethyl glycine units. Unlike DNA, which is highly negatively charged, the PNA backbone is neutral. Therefore, there is much less repulsive energy between complementary strands in a PNA-DNA hybrid than in the comparable DNA-DNA hybrid, and consequently they are much more stable. PNA can hybridize to DNA in either a Watson/Crick or Hoogsteen fashion (Demidov et al. Proc. Natl. Acad. Sci. U.S.A. 92:2637-2641, 1995; Egholm, Nature 365:566-568, 1993; Nielsen et al., Science 254:1497-1500, 1991; Dueholm et al., New J. Chem. 21:19-31, 1997). Molecules called PNA xe2x80x9cclampsxe2x80x9d have been synthesized which have two identical PNA sequences joined by a flexible hairpin linker containing three 8-amino-3,6-dioxaoctanoic acid units. When a PNA clamp is mixed with a complementary homopurine or homopyrimidine DNA target sequence, a PNA-DNA-PNA triplex hybrid can form which has been shown to be extremely stable (Bentin et al., Biochemistry 35:8863-8869, 1996; Egholm et al., Nucleic Acids Res. 23:217-222, 1995; Griffith et al., J. Am. Chem. Soc. 117:831-832, 1995).
The sequence-specific and high affinity duplex and triplex binding of PNA have been extensively described (Nielsen et al., Science 254.1497-1500, 1991; Eghohm et al., J. Am. Chem. Soc. 114:9677-9678, 1992; Egholm et al., Nature 365:566-568, 1993; Almarsson et al., Proc. Natl. Acad. Sci. U.S.A. 90:9542-9546, 1993; Demidov et al., Proc. Natl. Acad. Sci. U.S.A. 92:2637-2641, 1995). They have also been shown to be resistant to nuclease and protease digestion (Demidov et al., Biochem. Pharm. 48:1010-1313, 1994). PNA has been used to inhibit gene expression (Hanvey et al., Science 258:1481-1485,1992; Nielsen et al., Nucl. Acids. Res., 21:197-200, 1993; Nielsen et al., Gene 149:139-145, 1994), to block restriction enzyme activity (Nielsen et al., supra., 1993), to act as an artificial transcription promoter (Mollegaard, Proc. Natl. Acad. Sci. U.S.A. 91:3892-3895, 1994) and as a pseudo restriction endonuclease (Demidov et al., Nucl. Acids. Res. 21:2103-2107, 1993). Recently, PNA has also been shown to have antiviral and antitumoral activity mediated through an antisense mechanism (Norton, Nature Biotechnol., 14:615-619, 1996; Hirschman et al., J. Investig. Med. 44:347-351, 1996).
The ideal probe for irreversible chemical modification of plasmid will not dam the plasmid, and thus will not interfere with its transcription or intracellular trafficking. The plasmid structure, biological activity and stability would be the same with or without probe. The probe should be sequence-specific in order to differentiate delivered plasmid from endogenous nucleic acid and the probe itself should not have any influence on plasmid function. All of the technologies discussed above for chemically modifying plasmid DNA result in DNA damage and interfere with its transcriptional activity. Further, none of the technologies mentioned above allow direct detection of structurally and functionally intact plasmid in a real-time fashion on viable cells. The present invention provides a straightforward and versatile approach to permanently introduce new physical and biological properties into DNA by irreversible plasmid modification.
One embodiment of the present invention is a composition comprising a nucleic acid molecule and a conjugated peptide nucleic acid (PNA) molecule associated with said DNA molecule, wherein the PNA molecule contains a region complementary to the DNA molecule. In one aspect of this preferred embodiment, the nucleic acid molecule is DNA or RNA. Advantageously, the DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA molecule is a plasmid. In another aspect of this preferred embodiment, the plasmid encodes a reporter gene. Alternatively, the plasmid encodes a therapeutic gene. Advantageously, the reporter gene is xcex2-galactosidase, luciferase, chloramphenicol acetyltransferase green fluorescence protein or secreted alkaline phosphatase. Preferably, the PNA is conjugated to a fluorescent, colorimetric, radioactive or enzymatic label. In another aspect of this preferred embodiment, the PNA is conjugated to a protein, peptide, carbohydrate moiety or receptor ligand. Preferably, the peptide is a nuclear localization signal peptide, endosomal lytic peptide, transcriptional activator domain peptide, receptor specific peptide or immunostimulatory peptide.
The present invention also provides a method for determining the biodistribution of exogenous transfected nucleic acid molecule in a cell, comprising the steps of: contacting the exogenous nucleic acid molecule with a conjugated PNA in a sequence-specific manner prior to transfection; transfecting the cell with the labeled PNA; and monitoring the intracellular location of the nucleic acid molecule. In one aspect of this preferred embodiment, the nucleic acid molecule is DNA or RNA. Advantageously, the DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA is a plasmid. Advantageously, the PNA is conjugated to a fluorescent, colorimetric, radioactive or enzymatic label. In another aspect of this preferred embodiment, the transfecting step is mediated by cationic lipids.
Still another embodiment of the present invention is a method for enhancing the delivery of exogenous transfected nucleic acid molecule into the nuclear compartment of a cell, comprising the step of hybridizing the exogenous nucleic acid molecule to a PNA conjugated to a nuclear localization signal peptide prior to transfection. In one aspect of this preferred embodiment, the nucleic acid molecule is DNA or RNA. Advantageously, the DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA is plasmid DNA. Advantageously, the transfection is mediated by cationic lipids. In another aspect of this preferred embodiment, the nuclear localization signal peptide is poly-L-lysine, SV40 NLS, antennapedia peptide, TAT peptide, c-myc peptide, VirD2 peptide, nucleoplasmin peptide, ARNT derived peptide or M9 domain peptide.
The present invention also provides a method for promoting transcription of exogenous transfected DNA in a cell, comprising the step of hybridizing the exogenous DNA to a PNA conjugated to a transcription activator domain peptide prior to transfection. Advantageously, the exogenous transfected DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the exogenous transfected DNA is plasmid DNA. Advantageously, the transfection is mediated by cationic lipids. In one aspect of this preferred embodiment, the tanscription activator domain peptide is VP16 (337-347)2, P65 (520-550), Oct-2 (143-160), Sp1 (340-385), random acidic sequences or ERM (33-52).
Yet another embodiment of the present invention is a method for preventing entrapment of exogenous transfected nucleic acid molecule in the endosomal compartment of a cell, comprising the step of hybridizing the exogenous nucleic acid molecule to a PNA conjugated to an endosomal lytic peptide prior to transfection. In one aspect of this preferred embodiment, the nucleic acid molecule is DNA or RNA. Advantageously, the DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA is plasmid DNA. Advantageously, the transfection is mediated by cationic lipids. In another aspect of this preferred embodiment, the endosomal lytic peptide is HA derived peptide, GALA, KALA, EALA, melittin-derived peptide, xcex1-helical peptide or Alzheimer xcex2-amyloid peptide.
The present invention also provides a method for increasing the transfection efficiency of a transfected nucleic acid molecule in a cell, comprising the step of hybridizing the exogenous nucleic acid molecule to a PNA conjugated to a receptor specific ligand prior to transfection. In one aspect of this preferred embodiment, the nucleic acid is DNA or RNA. Advantageously, the DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA is plasmid DNA. Advantageously, the transfection is mediated by cationic lipids. In another aspect of this preferred embodiment, the receptor specific ligand is a sugar, immunoglobulin, IGF-1 derived peptide, xcex1V-integrin, epidermal growth factor, asialoglycoprotein, folate, transferrin or xcex12-macroglobulin.
Another embodiment of the invention is a method for screening compounds which activate transcription, comprising the steps of: linking a compound to a PNA; hybridizing a plasmid encoding a reporter gene to the PNA containing said linked compound; transfecting a cell with the plasmid-PNA-compound complex; determining the level of expression of the reporter gene; and comparing the level of expression of the reporter gene to the level of expression of the reporter gene in a cell transfected with the plasmid-PNA complex, wherein an increase in reporter gene expression in the presence of the compound indicates that the compound is an activator of transcription. Preferably, the reporter gene is xcex2-galactosidase, luciferase, chloramphenicol acetyltransferase green fluorescence protein or secreted alkaline phosphatase.
The present invention also provides a method for screening compounds which promote cellular uptake of an exogenous nucleic acid molecule, comprising the steps of linking a compound to a PNA; hybridizing a nucleic acid molecule to the PNA containing the linked compound; transfecting a cell with the nucleic acid-PNA-compound complex; determining the intracellular amount of the nucleic acid molecule; and comparing the intracellular level of the nucleic acid molecule to a cell transfected with a control complex not containing the compound, wherein an increase in the amount of the nucleic acid molecule in the cell compared to the control cell indicates that the compound promotes cellular uptake of the exogenous nucleic acid molecule. In one aspect of this preferred embodiment, the nucleic acid molecule is DNA or RNA. Advantageously, the DNA is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA is plasmid DNA.
Another embodiment of the present invention is a kit, comprising: a plasmid comprising a PNA binding site and a multiple cloning site for insertion of a nucleic acid sequence; a labeled PNA capable of binding to the PNA binding site; and sequencing primers complementary to the multiple cloning site. The kit may further comprise a labeling buffer. Preferably, the PNA is fluorescently labeled. Alternatively, the PNA is labeled with a chemical group capable of reacting with a chemical group on a protein. Preferably, the chemical group is pyridyldithiol or maleimide.
The present invention also provides a method for enhancing the immunogenicity of a protein or peptide encoded by an exogenous transfected DNA molecule, comprising the step of hybridizing the exogenous DNA molecule to a PNA conjugated to an immunostimulatory molecule prior to transfection. Preferably, the immunostimulatory molecule is a lymphokine, cytokine, muramyl dipeptide, complement-derived peptide or oligonucleotide. Advantageously, the oligonucleotide is a CpG dinucleotide repeat. Advantageously, the exogenous transfected DNA molecule is linear double stranded DNA, linear single stranded DNA, circular double stranded DNA or circular single stranded DNA. Preferably, the DNA is plasmid DNA.